Dysregulated pH is a hallmark of nearly all cancers regardless of tissue origin or genetic background. In normal cells, extracellular pH (pHe) is higher than intracellular pH (pHi) but in cancer cells this pH gradient is reversed. While the constitutive increase in pHi is small, it produces substantial changes in cell function. For example, the the increased pHi of cancer cells promotes cell proliferation and invasion while inhibiting apoptosis and altering cell metabolism. Here, we propose to investigate somatic mutations in cancer that may be conferring an increased fitness for the dysregulated pH of cancer. While somatic mutations in cancer are random, the increased pHi of cancers could provide a selective pressure for mutations that confer an adaptive advantage. Our hypothesis is that this selective pressure causes the conservation of histidine mutations in cancer that confer a gain in pH sensitivity. To investigate this hypothesis, in Aim 1 we will determine whether commonly occurring Arg>His mutations in candidate proteins confer a gain in pH-dependent function and what the mechanism of pH sensing might be. For each candidate we will test predictions that mutant but not wild- type proteins have pH-dependent functions by using biochemical assays in vitro and in cells. We also will test mechanisms of pH sensing of mutants by using molecular dynamics simulations and computational pKa predictions. In Aim 2 we will use database analyses to identify additional candidates with recurring histidine mutations, determine their frequency relative to codon bias frequency, and generate interaction networks to predict how these mutations might be conferring a functional adaptive advantage to cancer cells. These objectives will be achieved by using a database of amino acid mutations generated for the sponsor's laboratory from the Catalogue Of Somatic Mutations In Cancer (COSMIC) and established bioinformatics programs. To our knowledge this is the first examination of the adaptive retention and pH-dependent function of somatic mutations involving histidines in cancer. Our findings will generate new insights into how somatic mutations might be linked to the dysregulated pH in cancer. The results of this work might also reveal broad design principles of pH sensors that could inform the development of cancer therapeutics targeting pH-dependent pathways, regulatory mechanisms, or driver mutations.